Preparation of nucleic acids for electron microscopy normally requires metal staining or shadowing. To provide more quantitative images, we have developed a technique based on inelastic dark-field imaging in an energy-filtering transmission electron microscope to observe the native DNA directly. Microliter drops containing pUC8 plasmids that had been treated with spermidine to balance the strong negative charge of the DNA were applied to ultrathin (<3 nm) carbon films supported on lacy carbon/Formvar supports. After careful washing, the specimens were imaged by energy-filtering at low electron dose in the plasmon energy-loss range from 10-30 eV. Micrographs were recorded digitally at 120 keV beam energy by means of a cooled slow-scan CCD camera coupled to a single crystal YAG scintillator. Very clear images of the unstained and unshadowed DNA plasmids were obtained with a contrast level of approximately 30% above background. Imaging unstained macromolecules (DNA and proteins) with the inelastic dark-field signal has a significant advantage over conventional elastic dark-field imaging because there is approximately three times the amount of inelastic scattering than elastic scattering. This is a continuation of Intramural Research Project Z01-RR-10471-03 BEI.